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US20060281820A1 - Lfa-1 inhibitors and use thereof - Google Patents

Lfa-1 inhibitors and use thereof Download PDF

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US20060281820A1
US20060281820A1 US10/545,024 US54502403A US2006281820A1 US 20060281820 A1 US20060281820 A1 US 20060281820A1 US 54502403 A US54502403 A US 54502403A US 2006281820 A1 US2006281820 A1 US 2006281820A1
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lfa
inhibitor
diamine
spermine
cells
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Kuniyasu Soda
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C211/13—Amines containing three or more amino groups bound to the carbon skeleton
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to LFA-1 inhibitors and use thereof.
  • the present invention relates to a selective function inhibitor of LFA-1 containing polyamines, a pharmaceutical composition containing the LFA-1 inhibitor and a method for the inhibition or the treatment of diseases comprising the use of the LFA-1 inhibitor and the pharmaceutical composition.
  • CD cell membrane differentiation antigen
  • the CD includes adhesion molecules necessary as extracellular matrix for cell-cell adhesion. It has been clarified that the adhesion molecules not only contribute to cell-cell adhesion but also have important functions of precisely regulating various reactions of the body such as development and immune response by their action on intracellular signal transduction systems.
  • LFA-1 composed of cell membrane differentiation antigens CD11a and CD18 has been shown to serve an important function in formation of inflammation. It has been clarified that LFA-1 particularly existing in immunocytes such as peripheral blood monocyte (lymphocyte, monocyte, and macrophage) has a central role in a process from the very early stage to progression of various inflammations.
  • LFA-1 molecules are factors, which play a central role in formation and progression of many diseases such as arteriosclerosis, rejection of graft in organ transplantation, autoimmune disease, and allergic disease (1: reference number, it is described in the back of this specification as reference).
  • the LFA-1 is highly expressed in cells responsible for immunity, such as lymphocyte, monocyte, macrophage, and granular leukocyte, and there usually exists an adhesion partner to be paired with the LFA-1, that is, another adhesion molecule selectively binding to the LFA-1 adhesion molecule.
  • the signal transduction into cells starts by adhesion of this pair of adhesion molecules, and the cells are activated.
  • the LFA-1 binds selectively to the adhesion molecule called CD54 (another name ICAM-1: Intercellular adhesion molecule-1) highly expressed in vascular endothelial cell etc (2).
  • ICAM-1 is called ligand of LFA-1.
  • immunocytes such as lymphocyte, monocyte, macrophage, and granular leukocyte (granulocyte) having LFA-1 on the surface are activated by adhesion to vascular endothelial cell and the like expressing the ligand ICAM-1, and the inflammation is caused in the tissues.
  • the inflammation in the tissues starts by this reaction, activation of other adhesion molecules and functional cell membrane differentiation antigen molecules is induced by production of mediator inducing various inflammations, and the inflammations are increased. Therefore, development and progression of the inflammation can be inhibited by decreasing expression levels of LFA-1 or by binding antibodies and functional molecules to this adhesion molecule and decreasing the adhesive function of the adhesion molecule (3, 4, 5). In contrast, the inflammation can also be increased by increasing the expression of the LFA-1 (6).
  • LFA-1 has an important role in antitumor activity exhibited by immunocytes. It has been well known that the immunocytes recognize and kill cancer cells by culturing the immunocytes together with the cancer cells in the same cell culture medium. When the function of LFA-1 is inhibited by adding antibodies to LFA-1 to this culture medium, a part of antitumor activity to the cancer cells is decreased (7).
  • LFA-1 is a complex of CD11a and CD18. While the involvement of LFA-1 in a variety of diseases described above has been revealed by documents listed below, it has been clarified that the function of LFA-1 can be inhibited by inhibiting the expression of CD11a and CD18 composing the LFA-1, particularly the expression of CD11a.
  • therapeutic agents have been developed actively in order to achieve the inhibition of onset and the treatment of diseases for which LFA-1 has already been found to have a central role in development and progression of the pathology (arteriosclerosis, rejection of graft, autoimmune disease (type I diabetes (insulin-dependent diabetes mellitus), thyroid disease, autoimmune arthritis, cerebrospinal peripheral neuritis or degenerative disease), and the like), the treatment and inhibition of allergic disease, the inhibition of ischemic reperfusion injury, the inhibition and reduction of progression of hypertensive nephropathy and diabetic retinopathy, etc, by inhibiting LFA-1 or CD11a composing the LFA-1.
  • arteriosclerosis arteriosclerosis, rejection of graft, autoimmune disease (type I diabetes (insulin-dependent diabetes mellitus), thyroid disease, autoimmune arthritis, cerebrospinal peripheral neuritis or degenerative disease), and the like
  • autoimmune disease type I diabetes (insulin-dependent diabetes mellitus), thyroid disease, autoimmune arthritis, cerebrospinal peripheral neuritis
  • autoimmune disease type I diabetes, Graves' disease (Basedow disease), Hashimoto disease, autoimmune arthritis, cerebrospinal peripheral neurodegenerative disease and the like), allergic disease, ischemic reperfusion injury, and diabetic retinopathy
  • administration of anti-LFA-1 antibodies or substances having anti-LFA-1 effects to animals having the same pathology as these human diseases makes the inhibition of the disease or the improvement of the symptoms possible.
  • statin drug the agent for the treatment of hyperlipemia
  • function inhibition and cell adhesion inhibition of LFA-1 by the agent for the treatment of hyperlipemia are only exerted in the high concentration, which is not able to exist in a human body.
  • the antibodies and the small molecules are materials not essentially existing in the natural world and are foreign bodies in the human body, and it has been pointed out that when they are used in the body, it is unknown what side effect appears (17).
  • LFA-1 LFA-1 binds directly to LFA-1 molecules from the outside of the cells and physically delete the adhesive function.
  • the function of LFA-1 is regulated through stimulation to the cell having this adhesion molecule, which triggers the transmission from the inside of the cell of information for increasing expression of this LFA-1 molecule.
  • a substance named chemokine secreted from immunocytes as required by the living body reacts with chemokine receptor existing on the surface of the cell having LFA-1, and a signal of LFA-1 activation is thereby sent from the inside of the cell.
  • LFA-1 is activated, and the adhesive function of the cell is enhanced.
  • treatment with an agent forcedly putting LFA-1 in a mold from the outside and allowing the LFA-1 to fall into an immobilized state completely blocks physiological reaction in the living body.
  • dangerous side effects might be caused unless extensive safety for humans is confirmed.
  • mice not expressing LFA-1 in the cells are produced by gene manipulation, transfer of the tumor cell transplanted in this mouse is facilitated more than that of normal mice (18).
  • the present inventor has conducted studies in consideration of said circumstances and has consequently gained findings that a polyamine existing in the natural world and being ingested together with foods by humans inhibits the expression of cell membrane differentiation antigens CD11a and CD18 and as a result, inhibits the function of an LFA-1 adhesion molecule.
  • the present invention has been completed based on the findings.
  • An object of the present invention is to provide an LFA-1 inhibitor comprising a polyamine, a pharmaceutical composition comprising the LFA-1 inhibitor, and a method for the inhibition and the treatment of diseases with the LFA-1 inhibitors and the pharmaceutical compositions.
  • a further object of the present invention is to provide a selective function inhibitor of LFA-1 containing polyamines, a pharmaceutical composition for the treatment of arteriosclerosis comprising the LFA-1 inhibitors, a pharmaceutical composition for the inhibition of rejection comprising the LFA-1 inhibitors, a pharmaceutical composition for the treatment of autoimmune disease comprising the LFA-1 inhibitors, a pharmaceutical composition for the treatment of allergy comprising the LFA-1 inhibitors, a pharmaceutical composition for the treatment of ischemic reperfusion injury comprising the LFA-1 inhibitors, and a pharmaceutical composition for the treatment of diabetic retinopathy comprising the LFA-1 inhibitors.
  • the present invention provides the following invention.
  • the present invention provides an LFA-1 inhibitor comprising at least one member selected from the group consisting of polyamines having 2 to 6 amino groups and one or more linear or branched alkylene moieties having 2 to 7 carbon atoms and pharmaceutical acceptable salts thereof.
  • the present invention provides an LFA-1 inhibitor comprising at least one member selected from the group consisting of polyamines represented by the following general formula (1) and pharmaceutical acceptable salts thereof: NH 2 —(CH 2 )m1-(NH)p1(CH 2 )m2-(NH)p2-(CH 2 )m3-(NH)p3-(CH 2 )m4-(NH)p4-(CH 2 )m5-NH 2 (1) wherein each of m1 to m5 is independently an integer of 0 to 7, at least two of m1 to m5 exceed 0, the sum of m1+m2+m3+m4+m5 is 2 or more but less than 18, at least one of p1, p2, p3 and p4 is 1, and each of others independently represents 0 or 1.
  • polyamines represented by the following general formula (1) and pharmaceutical acceptable salts thereof: NH 2 —(CH 2 )m1-(NH)p1(CH 2 )m2-(NH)p2-(CH 2 )m3-(
  • the present invention further provides said LFA-1 inhibitor wherein the polyamine is selected from the group consisting of 3,3′-iminobispropylamine, N-aminobutyl-1,3-diaminopropane, 4,4′-iminobisbutylamine and N-aminopentyl-1,3-diaminopropane.
  • the polyamine is selected from the group consisting of 3,3′-iminobispropylamine, N-aminobutyl-1,3-diaminopropane, 4,4′-iminobisbutylamine and N-aminopentyl-1,3-diaminopropane.
  • the present invention further provides said LFA-1 inhibitor wherein the polyamine is selected from the group consisting of 4,9-diazatridecane-1,13-diamine, 4,9-diazadodecane-1,12-diamine, 4,8-diazadodecane-1,12-diamine, 5,9-diazatridecane-1,13-diamine, 4,9-diazatridecane-1,13-diamine, 4,10-diazatridecane-1,13-diamine, 4,9-diazatridecane-1,13-diamine, 5,9-diazatridecane-1,13-diamine and 5,9-diazatridecane-1,14-diamine.
  • the polyamine is selected from the group consisting of 4,9-diazatridecane-1,13-diamine, 4,9-diazadodecane-1,12-diamine, 4,8-diazadodecane-1,12-di
  • the present invention further provides said LFA-1 inhibitor wherein the polyamine is selected from the group consisting of 4,8,12-triazapentadecane-1,15-diamine, 4,8,12-triazahexadecane-1,16-diamine, 4,9,13-triazaheptadecane-1,17-diamine, 4,9,14-triazaoctadecane-1,18-diamine, 5,9,13-triazaheptadecane-1,17-diamine, 5,9,14-triazaoctadecane-1,18-diamine, 4,9,14-triazaoctadecane-1,18-diamine, and 5,10,14-triazaoctadecane-1,18-diamine.
  • the polyamine is selected from the group consisting of 4,8,12-triazapentadecane-1,15-diamine, 4,8,12-triazahexadecane-1,16-diamine, 4,9,13-triaza
  • the present invention further provides said LFA-1 inhibitor wherein the polyamine is selected from the group consisting of 4,8,12,16-tetraazanonadecane-1,19-diamine, 4,8,12,16-tetraazaicosane-1,20-diamine, 4,8,12,17-tetraazaicosane-1,20-diamine and 4,8,12,17-tetraazaicosane-1,20-diamine.
  • the polyamine is selected from the group consisting of 4,8,12,16-tetraazanonadecane-1,19-diamine, 4,8,12,16-tetraazaicosane-1,20-diamine, 4,8,12,17-tetraazaicosane-1,20-diamine and 4,8,12,17-tetraazaicosane-1,20-diamine.
  • the present invention further provides a pharmaceutical composition for the treatment of arteriosclerosis, a pharmaceutical composition for the inhibition of rejection, a pharmaceutical composition for the treatment of autoimmune disease, a pharmaceutical composition for the treatment of allergy, a pharmaceutical composition for the treatment of ischemic reperfusion injury, and a pharmaceutical composition for the treatment of diabetic retinopathy comprising said LFA-1 inhibitor.
  • the present invention further provides a method for the treatment of diseases selected from the group consisting of arteriosclerosis, autoimmune disease, allergy, ischemic reperfusion injury, and diabetic retinopathy comprising administrating said LFA-1 inhibitor.
  • the present invention further provides a method for the inhibition of diseases selected from the group consisting of arteriosclerosis, autoimmune disease, allergy, ischemic reperfusion injury, and diabetic retinopathy comprising administrating said LFA-1 inhibitor.
  • the present invention further provides a method for the inhibition of rejection characterized by comprising administrating said LFA-1 inhibitor.
  • polyamine means a compound containing three or more amino groups and two or more linear or branched alkylene moieties having 2 to 7 carbon atoms in the same molecule.
  • pharmaceutical acceptable salts means nontoxic acid addition salts of mineral acid or organic acid that can be used pharmaceutically.
  • patient means warm-blooded animals such as mammals of interest of treatment. Examples of animals that fall within the scope of the patient include dogs, cats, rats, house mice, horses, cattle, sheep, and humans.
  • CD11a is called LFA-1 ⁇ -chain, gp180/95, ⁇ L Integrin or the like as another name, and these names are unified into the name CD11a in the present specification and claims.
  • CD18 is called LFA-1 ⁇ -chain, Integrin ⁇ 2 or the like as another name, and these names are unified into the name CD18 in the present specification and claims.
  • FIG. 1 shows change (decrease) of average fluorescence intensity (measurement value) of CD11a of human peripheral blood monocyte cultured in cell culture medium with added spermine for 70-80 hours;
  • FIG. 2 shows change of CD11a histogram depending on spermine. It is shown that the number of the cells strongly expressing CD11a is decreased;
  • FIG. 3 shows change (decrease) of average fluorescence intensity of the cells expressing CD18 in the presence of spermine
  • FIG. 4 shows change (decrease) of average fluorescence intensity (measurement value) of CD11a of human peripheral blood monocyte cultured in cell culture medium with added spermine, spermidine or putrescine for 70-80 hours;
  • FIG. 5 shows that average fluorescence intensity of CD11a of peripheral blood monocyte when cultured with spermine for 20-26 hours has no change
  • FIG. 6 shows change (decrease) of average fluorescence intensity of CD11a of human peripheral blood monocytes which after cultured for 16-24 hours, were washed to remove spermine present outside of the cells, followed by culture in culture medium without spermine for 48-56 hours;
  • FIG. 7 shows change of average fluorescence intensity of the cells expressing adhesion molecules by spermine
  • FIG. 8 shows change of average fluorescence intensity of functional cell membrane differentiation antigen except adhesion molecules by spermine
  • FIG. 9 shows expression rate of CD11a positive cell and average fluorescence intensity of CD11a immediately after collection of blood and after culture for 72 hours;
  • FIG. 10 shows the rate (expression positive rate) to the total cells of cells that expressed adhesion molecules.
  • the expression rate of adhesion molecules of the cells cultured with spermine was not decreased;
  • FIG. 11 shows the rate (expression positive rate) to the total cells of the cells that expressed functional cell membrane differentiation antigen except adhesion molecules. The expression rate of cell membrane differentiation antigen of the cells cultured with spermine was not decreased;
  • FIG. 12 shows change of average fluorescence intensity depending on spermine in the experiment using peripheral blood monocyte accidentally infected during culture or in the body before collection of blood.
  • the expression of the cell membrane differentiation antigen such as CD16, 31, 49d, and 54 which is not usually decreased, is markedly decreased in the cells cultured with spermine;
  • FIG. 13 shows change of adhesive rate to culture plate of peripheral blood monocyte in the presence of spermine, spermidine, or putrescine
  • FIG. 14 shows a result of increasing, by centrifugation of culture plate, inhibition effect on cell adhesion to the culture plate by spermine
  • FIG. 15 shows that inhibition effect on adhesion to culture plate of the cells cultured with spermine for 20-24 hours is not apparent;
  • FIG. 16 shows change of adhesive (rate) to vascular endothelial cells of peripheral blood monocytes cultured with spermine for 20 hours or 72 hours. The inhibition effect on adhesion of the cells is not recognized in the culture for approximately 20 hours;
  • FIG. 17 shows change of the number of adhesive cells (actual number) to vascular endothelial cells of human peripheral blood monocytes which after cultured with spermine for 16-24 hours, were washed to remove spermine present outside of the cells, followed by culture in culture medium without spermine for 48-56 hours;
  • FIG. 18 shows change of adhesive (rate) to vascular endothelial cells of peripheral blood monocytes cultured with spermine, spermidine, or putrescine for 70-80 hours;
  • FIG. 19 shows change of cellular cytotoxicity and blastogenesis depending on spermine.
  • FIG. 20 shows change of polyamine concentration in peripheral blood monocyte cultured with polyamines (spermine, spermidine, or putrescine).
  • Polyamines used in the present invention are compounds having 3 to 6 amino groups and two or more linear or branched alkylene moieties having 2 to 7 carbon atoms.
  • the polyamines include compounds having the following chemical formula (1): NH 2 —(CH 2 )m1-(NH)p1-(CH 2 )m2-(NH)p2-(CH 2 )m3-(NH)p3-(CH 2 )m4-(NH)p4-(CH 2 )m5-NH 2 (1) wherein at least two of m1 to m5 exceed 0, each of m1 to m5 is independently an integer of 0 to 7, preferably an integer of 0 to 5, the sum of m1+m2+m3+m4+m5 is 2 or more but less than 18, preferably 2 or more but less than 17, more preferably 4 or more but less than 16, at least one of p1, p2, p3 and p4 is 1, and each of others independently represents 0 or 1.
  • Polyamines of the present invention include a compound wherein m1 and m2 are an integer of 2 to 7, in particular an integer of 3 to 5, m3, m4 and m5 are 0, p1 is 1, and p2, p3 and p4 are 0 in the formula (1).
  • Polyamines of the present invention include a compound wherein m1, m2 and m3 are an integer of 2 to 7, in particular an integer of 3 to 5, m4 and m5 are 0, p1 and p2 are 1, and p3 and p4 are 0 in the formula (1).
  • Polyamines of the present invention include a compound wherein m1, m2, m3 and m4 are an integer of 2 to 7, in particular an integer of 3 to 5, m5 is 0, p1 to p3 are 1, and p4 is 0 in the formula (1).
  • the polyamines of the present invention include triamine, tetraamine, pentaamine and hexamine, and can be used alone or in combination.
  • the polyamines of the present invention are specifically described below. References in patenthesis [ ] following each of the compounds are references describing a process for the production therefor.
  • Examples of the triamines used in the present invention include the followings:
  • the most preferable triamine is spermidine among the above.
  • tetraamines used in the present invention include the following:
  • Examples of pentaamines used in the present invention include the followings:
  • the preferable pentaamines are caldopentamine and homocaldopentamine.
  • hexaamines used in the present invention include the followings:
  • the preferable hexaamines are caldohexamine and homocaldohexamine.
  • said polyamines may be used in the pharmaceutically acceptable salt.
  • the salts may be addition salts of organic acid or mineral acid, and include, for example, mineral addition salts such as hydrochloric acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and for example, organic addition salts such as sulfonic acid, methansulfonic acid, sulfamic acid, tartaric acid, fumaric acid, hydrobromic acid, glycolic acid, citric acid, maleic acid, phosphoric acid, succinic acid, acetic acid, benzoic acid, ascorbic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, propionic acid, lactic acid, pyruvic acid, oxalic acid, stearic acid, cinnamic acid, aspartic acid, salicylic acid, and gluconic acid.
  • mineral addition salts such as hydroch
  • the salts are advantageous in the treatment because those are fume-free of free base.
  • hydrochloric acid addition salt is the most preferable.
  • the acid addition salts can be easily prepared by contact of polyamines of free base form with suitable acid.
  • a dose of the LFA-1 inhibitor of the present invention is changed appropriately according to route of administration, and sex, symptoms, age and body weight of the patient. Generally, the dose is 0.01-100 mg/Kg of body weight, in particular 0.05-40 mg/kg of body weight, and more preferably 0.05-4 mg/kg of body weight as the polyamines, per day for a human adult.
  • said polyamines or combinations thereof as active ingredients can be used alone or in combination with other desired agents.
  • the LFA-1 inhibitors or the pharmaceutical compositions of the present invention can be administered orally or parenterally.
  • the form of the parenteral administration includes administration by injection such as instillation, intravenous injection, hypodermic injection, and intramuscular injection, percutaneous administration with ointment and percutaneous agents, and rectal administration with suppository.
  • the LFA-1 inhibitors or the pharmaceutical compositions of the present invention can be prepared in the form of hard capsule, soft capsule, granule, powder, subtle granule, ball, troche, active ingredient sustained release agent, solution, suspension and the like. The preparation can be carried out easily by using general carrier of pharmaceutical field and by conventional means.
  • compositions for the preparation such as general carrier, for example diluent and excipient such as filler, expander, binder, disintegrating agents, surfactant, and lubricant, can be used.
  • general carrier for example diluent and excipient such as filler, expander, binder, disintegrating agents, surfactant, and lubricant
  • parenteral administration of the pharmaceutical compositions of the present invention can be prepared by dissolving said polyamines alone or together with other compositions for the preparation in an appropriate solvent such as saline and phosphate buffer solution.
  • Polyamine is low molecular basic material containing nitrogen atoms and named from containing amine moieties in the molecule. Polyamine is contained in cells of almost all organisms including microorganisms, plants and animals at high concentration, i.e. mM unit (M represents mol/L). It is believed that polyamine plays a key role in cell proliferation or differentiation and intracellular signaling. It had been clarified that intracellular polyamine concentration in young individual which cell proliferation is active is high, and intracellular polyamine concentration is rapidly decreased by aging (20, 21). This is considered to be mainly because activity of intracellular enzyme for synthesizing polyamine described below is rapidly decreased with aging (22).
  • polyamines in the human body are mainly three kinds, spermine, spermidine and putrescine.
  • polyamines are synthesized in the cell, and their synthetic pathways have been elucidated fully.
  • ornithine is synthesized from arginine which is a kind of amino acid, by the effects of arginase.
  • Ornithine is converted into putrescine by the effects of ornithine decarboxylase. From the production of the putrescine, the synthesis of polyamine starts.
  • S-adenosylmethionine synthesized from methionine is decarboxylated by the effects of S-adenosylmethionine decarboxylase, and decarboxylated S-adenosylmethionine is produced.
  • spermidine synthase another name: spermine-spermidine synthase
  • the putrescine undergoes propylamine transfer from the decarboxylated S-adenosylmethionine, and spermidine is synthesized.
  • spermine synthase or spermine-spermidine synthase
  • spermidine undergoes propylamine transfer from the decarboxylated S-adenosylmethionine, and spermine is synthesized.
  • the spermine is acetylated and converted into N-acetylspermine by the effects of AcetylCoA, and further converted into spermidine by the effects of polyamine oxidase.
  • spermidine is acetylated and converted into N-acetylspermidine by the effects of acetylCoA, and further converted into putrescine by the effects of polyamine oxidase.
  • spermine and spermidine are synthesized and decomposed in the cell by the effects of synthetic enzyme and the effects of catabolic enzyme, and the intracellular concentrations of spermine and spermidine are adjusted.
  • Polyamine concentration in blood as well as excretion of polyamines into urine is increased in diseases which actively produce polyamines.
  • the excretion of polyamines into urine requires transporting the polyamines to kidney though blood flow.
  • polyamines are not present in plasma, it is considered that blood cells are responsible for the transportation of the polyamines in the body.
  • polyamines are hardly detected in human plasma, and most of polyamines detected in blood are contained in erythrocyte. This is because the number of erythrocyte is large, in spite of low polyamine concentration in the erythrocyte.
  • lymphocyte spermidine and spermine are contained in concentrations 100 times and 400 times, respectively, as much as their concentrations in erythrocyte.
  • spermidine is contained in concentration higher than that of spermine.
  • lymphocyte and granular leukocyte spermine is contained in concentration higher than that of spermidine (24).
  • polyamines move in the body by cells in blood (erythrocyte, leukocyte, lymphocyte, monocyte, and macrophage).
  • intracellular polyamine concentration is adjusted by synthesis and decomposition of polyamines in the cell, uptake thereof from the outside of the cell, and excretion thereof to the outside of the cell.
  • Spermidine and spermine ingested orally have a significant impact on the concentration and construction of polyamines in the cells (including peripheral blood monocyte (lymphocyte, monocyte, and macrophage)) in the body.
  • polyamines are contained in foods.
  • Polyamine concentration and component ratio of polyamines differ largely from food to food.
  • the contents of spermine and spermidine which are absorbed in their original molecular forms and taken up into the cell, differ largely depending on food types.
  • Spermine and spermidine are contained in high concentration in some beans such as soybean and green pea and mushrooms among natural foods.
  • Large amounts of spermine and spermidine are also contained in fermented food products such as cheese and yogurt among processed foodstuffs (30).
  • the type and amount of polyamines (particularly, spermine and spermidine) ingested by humans in a day differ largely depending on differences of eating habits in the regions where they live.
  • Possible methods for increasing intracellular spermine and spermidine concentrations include: a method comprising administering putrescine, ornithine, arginine, methionine, and S-adenosylmethionine serving as raw materials of spermine and spermidine; a method comprising activating enzymes necessary for intracellular synthesis of spermidine and spermine (e.g., ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, spermine synthase, and spermine-spermidine synthase) and promoting synthesis of spermine and spermidine; a method comprising inhibiting the activity of enzymes which decompose the synthesized spermine and spermidine in the cell; and a method comprising orally or parenterally administering spermine and spermidine to allow them to be directly taken up form the outside of the body into the cell.
  • the method comprising orally or parenterally administering spermine and spermidine is simple and is capable of effectively increasing intracellular spermine and spermidine concentrations.
  • previous studies have also elucidated the daily intake of polyamines in human and the amounts of spermine and spermidine necessary to express acute toxicity.
  • the reverse effects of the methods described above may be employed. Namely, it is possible to decrease intracellular spermine and spermidine concentrations by a method comprising cutting off supply of putrescine, ornithine, arginine, methionine, S-adenosylmethionine and the like serving as substances necessary for synthesis of spermine and spermidine; a method comprising inhibiting the effects of enzymes necessary for synthesis of spermidine and spermine (e.g., ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, spermine synthase, and spermine-spermidine synthase); a method comprising activating acetylCoA and polyamine oxidase having the effects of decomposing spermine and spermidine and promoting the decomposition of spermine and spermidine; and
  • biogenic polyamines are inherently present in organisms, and the intracellular concentration thereof can be adjusted.
  • polyamine concentration in an organism is adjusted in uses for inhibiting the expression of CD11a and CD18 and inhibiting the function of LFA-1 composed of both of them, as in the present invention.
  • a phenomenon in which the function of LFA-1 is directly inhibited is a novel finding gained by the present inventor.
  • a pharmaceutical composition containing the LFA-1 inhibitor of the present invention has an effect on the treatment and inhibition of diseases such as arteriosclerosis, autoimmune disease, allergy, ischemic reperfusion injury, and diabetic retinopathy, via the expression level of LFA-1 and the strong action of inhibiting LFA-1.
  • diseases such as arteriosclerosis, autoimmune disease, allergy, ischemic reperfusion injury, and diabetic retinopathy.
  • the improvement, treatment, and inhibition effects on their symptoms are illustrated below.
  • a pharmaceutical composition for the treatment of arteriosclerosis containing said LFA-1 inhibitor of the present invention and a method for the treatment and the inhibition of arteriosclerosis using the pharmaceutical composition are described.
  • a preparation form, a method for the preparation, and a method for the administration of the pharmaceutical composition are the same as said LFA-1 inhibitor.
  • a dose for the treatment of the disease that occurred is 0.02-20 mg/kg of body weight per day, preferably 0.05-10 mg/kg of body weight per day.
  • the polyamine is administered daily.
  • a dose for the inhibition of onset and progression of arteriosclerosis is 0.05-4 mg/kg of body weight per day.
  • LFA-1 inhibitor which inhibits the expression of CD11a and CD18 and inhibits the function of LFA-1, are described based on documents.
  • pravastatin which is a drug of hyperlipemia and also serves as an LFA-1 inhibitor inhibits arteriosclerosis of coronary artery (nutritional blood vessel of heart) of a human patient after cardiac transplantation (31).
  • statin-based therapeutic agent of hyperlipemia inhibits arteriosclerosis, and its mechanism works through the inhibition of LFA-1 by the statin-based therapeutic agent (32).
  • lovastatin which is a therapeutic agent of hyperlipemia blocks adhesion between LFA-1 and ICAM-1 and inhibits arteriosclerosis (33).
  • atorvastatin which is a therapeutic agent of hyperlipemia inhibits monocyte adhesion to vascular endothelial cell, and have indicated that this is the mechanism of arteriosclerosis inhibition (34).
  • a pharmaceutical composition for the treatment of autoimmune disease containing said LFA-1 inhibitor of the present invention and a method for the treatment and the inhibition of autoimmune disease using the pharmaceutical composition are described.
  • a preparation form, a method for the preparation, and a method for the administration of the pharmaceutical composition are the same as said LFA-1 inhibitor.
  • said polyamine is administered at 0.02 to 20 mg/kg of body weight per day, preferably 0.05 to 10 mg/kg of body weight per day.
  • said polyamine is administered at 0.05 to 4 mg/kg of body weight per day for consecutive days.
  • LFA-1 inhibitor which inhibits the expression of CD11a and CD18 and inhibits the function of LFA-1, are described based on documents.
  • autoimmune disease of interest of treatment with the pharmaceutical composition of the present invention examples include the following: psoriasis, type I diabetes (insulin-dependent diabetes mellitus), Graves' disease (Basedow disease), Hashimoto disease, autoimmune arthritis (Lyme arthritis, chronic rheumatoid arthritis), autoimmune cerebrospinal peripheral neuritis or degeneration, Sjogren's syndrome, uveitis, retinitis, degeneration, autoimmune renal disease (glomerulonephritis and the like), inflammatory bowel disease (Crohn disease, ulcerative colitis and the like), and primary cholangitis.
  • psoriasis type I diabetes (insulin-dependent diabetes mellitus), Graves' disease (Basedow disease), Hashimoto disease, autoimmune arthritis (Lyme arthritis, chronic rheumatoid arthritis), autoimmune cerebrospinal peripheral neuritis or degeneration, Sjogren's syndrome, uveitis, retinitis, degeneration, autoimmune
  • Gottschsch et al. have reported clinical examples that psoriasis is improved by administration of efalizumab which is a CD11a antibody (40, 41).
  • Mysliwiec et al. have indicated that enhancement of fluorescence intensity of CD11a positive cell in blood of peripheral blood monocyte of a patient with type I diabetes (insulin-dependent diabetes mellitus) and a patient with high risk of onset thereof is proportional to a detection level of autoantibody against langerhans islet (insulin-secreting cell), and have revealed that LFA-1 has an important role in onset of type I diabetes (44).
  • LFA-1 has the structure moiety similar to antigen of offending bacteria of Lyme arthritis, and may have a central role of the pathology (53).
  • LFA-1 molecules may be the target of autoimmunity in pathology of Lyme arthritis which is special arthritis (54).
  • Birner et al. have reported that infiltration of lymphocytes to joints is increased in arthritis rheumatica, and by administration of LFA-1 antibodies to rat with similar disease, the symptoms of the arthritis are reduced (55).
  • ICAM-1 which is LFA-1 ligand
  • Sjogren's syndrome which is autoimmune disease
  • LFA-1 is expressed highly in the lymphocytes infiltrated to lesion of the salivary glands (61).
  • Hayashi et al. have reported that when the disease of mice with pathology similar to Sjogren's syndrome is transplanted to other mice, the disease can be prevented by administration of anti-LFA-1 antibody (62).
  • Whitcup et al. have reported onset of uveitis can be inhibited by administration of antibodies against LFA-1 and ICAM-1 to rats with the same disease as uveitis in human (64).
  • Nishikawa et al. have reported that progression of nephritis is inhibited by administration of anti-LFA-1 antibodies to rats with disease similar to human glomerulonephritis (66).
  • Kootstra et al have reported that the finding of nephritis is improved by administration of anti-LFA-1 antibodies to animal model of lupus nephritis which is autoimmune disease in human (68).
  • Taniguchi et al have reported that by administration of anti-ICAM-1 antibodies to disease models of rats with the disease similar to human inflammatory bowel disease (ulcerative colitis and Crohn disease) and inhibition of adhesion to intestinal mucosa cells of immunocytes expressing LFA-1, the symptom is improved (69).
  • Vainer et al have reported that expression of CD18 is enhanced in immunocytes infiltrated in intestinal mucosa of patients with ulcerative colitis, and expression of CD11a is enhanced in the cell of patients with Crohn disease (70).
  • a pharmaceutical composition for the treatment of allergy containing said LFA-1 inhibitor of the present invention and a method for the treatment and the inhibition of allergy using the pharmaceutical composition are described.
  • a preparation form, a method for the preparation, and a method for the administration of the pharmaceutical composition are the same as said LFA-1 inhibitor.
  • said polyamine When used in the treatment of allergy that occurred, said polyamine is administered at 0.02 to 20 mg/kg of body weight per day, preferably 0.05 to 10 mg/kg of body weight per day.
  • said polyamine is administered at 0.05 to 4 mg/kg of body weight per day for consecutive days.
  • LFA-1 inhibitor which inhibits the expression of CD11a and CD18 and inhibits the function of LFA-1, are described based on documents.
  • Winquist et al. have reported that by administration of a small molecule produced for inhibition of LFA-1 to allergic dermatitis of animals, the symptom is improved (78).
  • Murayama et al. have reported that by administration of anti-LFA-1 antibodies to contact dermatitis of mice, the symptom is improved (78).
  • Bloemen et al. have reported that when allergic asthma is induced in a mouse, the mouse has resistance to the asthma by administration of anti-LFA-1 antibodies (80).
  • a pharmaceutical composition for the treatment of ischemic reperfusion injury containing said LFA-1 inhibitor of the present invention and a method for the treatment and the inhibition of ischemic reperfusion injury using the pharmaceutical composition are described.
  • a preparation form, a method for the preparation, and a method for the administration of the pharmaceutical composition are the same as said LFA-1 inhibitor.
  • said polyamine is administered at 0.02 to 40 mg/kg of body weight per day, preferably 0.05 to 20 mg/kg of body weight per day.
  • LFA-1 inhibitor which inhibits the expression of CD11a and CD18 and inhibits the function of LFA-1, are described based on documents.
  • Ischemic reperfusion injury refers to tissue injury caused by reperfusion of blood after vascular obstruction to tissues of cardiac infarction, angina pectoris, cerebral infarction, transient cerebral ischemic attack, transplanted organs, and the like, in which blood flow was transiently blocked for a certain period.
  • a pharmaceutical composition for the treatment of diabetic retinopathy containing said LFA-1 inhibitor of the present invention and a method for the treatment and the inhibition of diabetic retinopathy using the pharmaceutical composition are described.
  • a preparation form, a method for the preparation, and a method for the administration of the pharmaceutical composition are the same as said LFA-1 inhibitor.
  • said polyamine is administered at 0.02 to 20 mg/kg of body weight per day, preferably 0.05 to 10 mg/kg of body weight per day.
  • the inhibition of diabetic retinopathy and the improvement of the symptoms can be conducted by the LFA-1 inhibitor, which inhibits the expression of CD11a and CD18 and inhibits the function of LFA-1.
  • LFA-1 inhibitor which inhibits the expression of CD11a and CD18 and inhibits the function of LFA-1.
  • Barouch et al. have indicated that when the functions of LFA-1 are inhibited by administration of antibodies of CD18 to diabetic rats, the number of leukocytes infiltrated in retina is decreased, and have reported that the antibodies have the effects of inhibiting the progression of diabetic retinopathy (88).
  • a pharmaceutical composition containing LFA-1 inhibitors has an effect on the treatment and inhibition of diabetic retinopathy. Inhibition of rejection and method thereof.
  • a pharmaceutical composition containing said LFA-1 inhibitor of the present invention has inhibition effects on rejection in transplantation.
  • a preparation form, a method for the preparation, and a method for the administration of the pharmaceutical composition are the same as said LFA-1 inhibitor.
  • the inhibition of rejection of transplanted organs and tissues and the improvement of graft survival rate of the transplanted organs and tissues can be performed by the LFA-1 inhibitor, which inhibits the expression of CD11a and CD18 and inhibits the function of LFA-1.
  • said polyamine is administered at 0.02 to 20 mg/kg of body weight per day, preferably 0.05 to 10 mg/kg of body weight per day.
  • the perfusate containing 1 ⁇ M to 10 mM of said polyamine, preferably 10 ⁇ M to 2 mM of said polyamine is used.
  • pravastatin which is a drug of hyperlipemia and also serves as an LFA-1 inhibitor increases graft survival rate of transplanted heart in human patients of cardiac transplantation, and have reported this finding (89).
  • peripheral blood monocytes including lymphocyte, monocyte and the like were recovered from the collected blood by specific gravity centrifugation technique using SEPARATE-L (Muto Pure Chemicals Co. LTD., Tokyo, Japan).
  • SEPARATE-L Semangiol-L
  • the recovered peripheral blood monocytes were suspended in PRMI1640 culture medium (Sigma chemical co., St. Louis, USA) mixed with 10% human serum (Wako Pure Chemical Industries LTD., Osaka, Japan), 0.1% L-glutamine (Invitrogen Corp., CA, USA), and 0.01% penicillin-streptomycin (Invitrogen Corp., CA, USA) and cultured in the humidified air at 37° C. containing 5% carbonic acid gas.
  • spermine spermine tetrahydrochloride; ICN Biomedicals Inc., Ohio, USA
  • spermidine spermidine trihydrochloride; ICN Biomedicals Inc., Ohio, USA
  • putrescine 1,4-Butanediamine dihydrochloride; Wako Pure Chemical Industries LTD, Osaka, Japan
  • peripheral blood monocytes which after cultured with the polyamines for 16 to 24 hours, were washed and further cultured in culture medium without polyamines for 48 to 56 hours, were used as the different peripheral blood monocytes in Examples 2, 4, 5, and 6 below.
  • the peripheral blood monocytes cultured for 16 to 80 hours by the method of Example 1 were collected from the cell culture plate so as not to damage the cells.
  • the collected cells were washed with PBS ( ⁇ ) solution and then fixed using phosphate buffered saline without calcium chloride and without magnesium chloride (hereinafter, referred to as PBS ( ⁇ ); Invitrogen Corporation, GIBCO, Grand Island, N.Y., USA) containing 2% paraformaldehyde (Wako Pure Chemical Industries LTD, Osaka, Japan), so as not to achieve change in cell membrane antigen molecules on the cell surface of the peripheral blood monocytes.
  • PBS phosphate buffered saline without calcium chloride and without magnesium chloride
  • the peripheral blood monocytes were further washed, to which antibodies to the cell membrane antigen molecules were then added in an amount corresponding to 5 ⁇ L (microliter) per 500000 cells.
  • the antibodies used here are CD2 (FITC label), CD4 (FITC), CD8 (PE label), CD11a (FITC), CD11b (PE), CD11c (PE), CD18 (FITC), CD31 (PE), CD49d (PE), CD49e (PE), CD54 (PE), CD62L (PE), CD95 (FITC), and VIA-PROBE (FITC). All of these antibodies are manufactured by PharMingen (A Becton Dickinson Company, San Jose, Calif., USA).
  • the average fluorescence intensity of CD11a and CD18 among the cell membrane surface antigens of the human peripheral blood monocytes (including lymphocyte, monocyte, macrophage, and natural killer (NK) cell) cultured in the cell culture medium mixed with spermine was inhibited. This inhibition got stronger with increase of spermine concentration, and concentration dependence was observed in the inhibition of fluorescence intensity of CD11a by spermine. Namely, as described in FIG. 1 , the average fluorescence intensity of CD11a of the peripheral blood monocytes cultured with spermine for 70 to 80 hours was reduced as spermine concentration was increased. Each symbol in FIG. 1 represents an actual measurement value of change of average fluorescence intensity of CD11a in each individual blood.
  • CD11a histogram is shown in FIG. 2 .
  • the horizontal axis of the histogram represents fluorescence intensity of the cells. This means that a cell expressing CD11a more strongly is located closer to the right on the horizontal axis.
  • the vertical axis of the histogram represents the number of the cells. This means that a hill gets higher in the direction of the vertical axis as the number of the cells having the same fluorescence intensity gets larger. As seen from FIG. 2 , a right-hand hill in the histogram is low for the cells supplemented with spermine, indicating that the number of the cells having strong fluorescent intensity is decreased.
  • CD18 which is a molecule composing LFA-1 together with CD11a
  • spermine FIG. 3
  • the average fluorescence intensity of CD18 was decreased with increase of spermine concentration.
  • the average fluorescence intensity of CD11a of the human peripheral blood monocytes cultured in the cell culture medium mixed with spermidine was inhibited dependently on spermidine concentration. This decreasing effect was strongest in spermine, followed by spermidine, while the decreasing effect exhibited by putrescine was not evident.
  • the decrease of the average fluorescence intensity of CD11a is not derived from changes produced by the contact for a long time between high concentration of polyamines and the peripheral blood monocytes, that is, is not the direct effect by the extracellular polyamines on the CD11a molecule on the cell surface from the outside of the cells.
  • the average fluorescence intensity of adhesion molecules (CD11b, CD11c, CD31, CD49d, CD49e, CD54, and CD62L) except CD11a and CD18 was not decreased by spermine and spermidine.
  • the average fluorescence intensity of CD62L was obviously increased dependently on spermine concentration.
  • CD11a bright cells strongly expressing CD11a
  • CD11a bright expression of CD11a bright was supposed to be increased by stimulation of the culture plate but was not increased by the presence of spermine or spermidine.
  • FIG. 9 when comparison before and after cell culture was made on average fluorescence intensity of CD11a and expression rate of CD11a positive cell, it was clarified that the fluorescence intensity and the expression rate after the culture were decreased more than those before the culture.
  • the hypothesis was denied that expression intensity of CD11a was supposed to be increased by the stimulation of the peripheral blood monocytes with the culture plate but this increase was inhibited by the presence of spermine or spermidine.
  • the expression positive cells of the cell membrane differentiation antigens having an important role in cell functions, other than the adhesion molecules were not decreased.
  • VIA-Probe is taken up into dead cells but not into live cells and as such, can be used to investigate the number of the dead cells.
  • the dead cells were not increased.
  • spermine and spermidine exhibit no cellular cytotoxicity and selectively inhibit only CD11a and CD18 among the cell surface antigens.
  • the polyamines have an effect of inhibiting cytokine production (99).
  • cytokine is produced from the cultured cells by stimulation of virus and bacteria etc, but the production of the cytokine is inhibited by the polyamines. Meanwhile, expression of any of adhesion molecules is increased by the production of the cytokine (100, 101, 102, 103).
  • cytokine production should be inhibited with increase of polyamine concentration, and expression of the adhesion molecules whose expression is increased by the cytokine production should be decreased apparently.
  • Data of an experiment that accidentally caused viral or microbial infection is shown in FIG. 12 .
  • average fluorescence intensity of CD16, CD31, CD49d, and CD54 previously reported to have expression increased by cytokine is low in the cells cultured in the culture medium supplemented with the polyamines. This change is totally different from the change of the cell membrane differentiation antigens by the polyamines in the absence of infection.
  • the inhibition of average fluorescence intensity of CD11a and CD18 by the polyamines is not the inhibition by the polyamines of increase of the average fluorescence intensity by physical and chemical stimulation attributed to cell culture.
  • Peripheral blood monocytes were cultured on a 96-well cell culture plate using the culture medium described above. After spermine, spermidine, or putrescine was added in varying concentrations, the resulting cells were cultured for a given time. Following the culture, the cell culture plate was washed three times with PBS ( ⁇ ) solution to remove the cells floating in the culture medium. As a result, there remained only the cells adhering on the inside bottom of the cell culture plate.
  • the cells were cultured with varying concentrations of spermine for 70 to 82 hours. Then, the cell culture plate was inverted and centrifuged in a centrifuge at 500 rpm for 5 minutes to exfoliate the cells weakly adhering on the inside bottom of the culture plate. The culture supernatant was removed. As a result, there remained only the cells firmly adhering on the inside bottom of the cell culture plate, as described above.
  • MTT Thiazolyl blue
  • the cells were dissolved by addition of 100 ⁇ l of cell lysis solution (2-propanol (isopropanol; Wako Pure Chemical Industries LTD, Osaka, Japan) mixed with 12 M (mol/L) hydrochloric acid (HCl; Wako Pure Chemical Industries LTD, Osaka, Japan)), to recover the dye in the cells.
  • Absorbance in the cell culture plate was measured at 2 absorbance wavelengths of 570 nm and 690 nm using an absorbance reader (Titertek Multiskan MCC/340, Labsystems, Flow Laboratories Inc., USA).
  • peripheral blood monocytes cultured simultaneously with the culture described above under the same conditions were used to conduct an experiment for investigating the number and activity of the total cultured cells. Namely, MTT was added to the culture supernatant of the peripheral blood monocytes cultured for a given time in a culture plate supplemented with varying concentrations of spermine, spermidine, or putrescine, and the cells were cultured at 37° C. for 2 to 4 hours until the cells were sufficiently stained. After the cells were sufficiently stained, the culture plate was centrifuged in a centrifuge at 1000 rpm for 10 minutes to allow all the cells present in the culture plate to firmly adhere on the inside bottom of the plate.
  • the culture supernatant in the plate was removed with the utmost caution not to soak up the cells adhering on the inside bottom of the plate. This means that all the cells present in the cell culture plate exist on the inside bottom of the plate.
  • the dye in the cells was released by the addition of 100 ⁇ l of cell lysis solution (isopropanol mixed with 12 M (mol/L) hydrochloric acid) to the culture plate. Absorbance of the cell culture medium was measured at 2 absorbance wavelengths of 570 nm and 690 nm using an absorbance reader (Titertek Multiskan MCC/340, Labsystems, Flow Laboratories Inc., USA). As a result, each polyamine concentration and the total amount of live cells of the cultured peripheral blood monocytes can be confirmed.
  • the selective inhibition effect on the average fluorescence intensity of CD11a and CD18 by spermine and spermidine was not an apparent effect and was an effect of inhibiting the adhesive function, which is important function of the adhesion molecule LFA-1 composed of CD11a and CD18.
  • Example 2 Detection of cell membrane differentiation antigen of human peripheral blood monocyte. Namely, in the result of Example 2, it was clarified that the inhibition of average fluorescence intensity of CD11a and CD18 by spermine and spermidine was not exhibited in the culture for a time as short as approximately 24 hours.
  • Example 3 it was clarified that the inhibition of function of the LFA-1 molecule composed of CD11a and CD18, that is, the inhibition of cell adhesion to the culture plate also requires time as much as approximately 70 hours when the inhibition of expression of CD11a and CD18 was remarkable in Example 2.
  • MTT is taken up only into live cells and reflects cell activity.
  • no reduction in uptake of the MTT dye and discoloring of the dye in the peripheral blood monocytes cultured with high concentrations of spermine or spermidine also indicates that the cell activity and the number of the cells are not decreased. From this result, it is evident that spermine and spermidine in high concentrations (up to 1 mM) produce no cellular cytotoxicity to the peripheral blood monocytes. Because only the adhesive function of the cells was inhibited by spermine and spermidine, it is evident that the adhesion to the cell culture plate was suppressed.
  • LFA-1 molecules on the surface of peripheral blood monocytes bind to their ligand CD54 (ICAM-1) in vascular endothelial cells in the early stage of inflammation and in arteriosclerosis, and immunocytes are thereby stimulated.
  • IAM-1 ligand CD54
  • Monocyte and the like secrete various factors involved in inflammation by this stimulation, and inflammation gradually progresses. This mechanism occurs in the initial stage of inflammation and is an important reaction.
  • peripheral blood monocyte adhesion to vascular endothelial cells were cultured for 3 days in cell culture medium containing 0 ⁇ M, 100 ⁇ M, or 500 ⁇ M spermine, spermidine, or putrescine.
  • peripheral blood monocytes were cultured with spermine or spermidine for approximately 16 to 24 hours, then cells were recovered and washed three times with PBS ( ⁇ ) to remove the extracellular polyamines, followed by culture for 48 hours in polyamine-free RPMI1640 culture medium containing 10% human serum.
  • Vascular endothelial cells used were collected from the vein in umbilical cord from volunteers and subcultured on a culture plate. Techniques of collection of the vascular endothelial cells and methods for the preservation and subculture thereof have no direct bearing on the techniques of the present invention, and the description thereof is therefore omitted.
  • the endothelial cells of human umbilical cord were cultured in another cell culture plate and spread all over the culture plate (RPMI1640+10% fetal bovine serum was used in culture medium for this cell culture).
  • RPMI1640+10% fetal bovine serum was used in culture medium for this cell culture.
  • the internal environment of blood vessel could be reconstituted on the culture plate.
  • the adhesion between peripheral blood monocytes and vascular endothelial cells, which actually occurs in human or animal blood vessel can be observed by adding the peripheral blood monocytes to the culture plate having the inside bottom fully covered with the vascular endothelial cells.
  • peripheral blood monocytes which were cultured for 70 to 80 hours in cell culture medium (RPMI1640 supplemented with 10% human serum, 0.1% L-glutamine, and 0.01% penicillin-streptomycin) mixed with the polyamines, or peripheral blood monocytes which after cultured in the cell culture medium containing the polyamines for 16 to 24 hours, were washed with PBS ( ⁇ ) to remove the polyamines present in the cell culture medium, followed by culture in cell culture medium without polyamines for 48 to 56 hours were used.
  • cell culture medium RPMI1640 supplemented with 10% human serum, 0.1% L-glutamine, and 0.01% penicillin-streptomycin
  • the cultured peripheral blood monocytes were recovered from the culture plate and washed three times with PBS ( ⁇ ) solution, followed by adjustment of the number of the cells to 5 ⁇ 10 6 cells/ml.
  • the resulting cells were cultured for 1 hour in 5 ⁇ M cell culture medium (RPMI1640+10% fetal bovine serum) containing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Molecular Probes, Oregon, USA) (fluorescent reagent).
  • peripheral blood monocytes fluorescently labeled with BCECF-AM were respectively adjusted to 1 ⁇ 10 7 cells/ml, and 100- ⁇ L aliquot each thereof was mixed into the cell culture plate in which the vascular endothelial cells were cultured. Following additional culture at 37° C. for 30 minutes, the culture plate was filled with culture medium, then sealed, and inverted at room temperature for 30 minutes. By this procedure, there remain only the peripheral blood monocytes firmly adhering to the vascular endothelial cells covering the inside bottom of the culture plate, whereas nonadhering peripheral blood monocytes can be removed.
  • the culture medium was removed, and the cells were dissolved by addition of 50 ⁇ L of aqueous solution of 50 mM Tris-HCl (Wako Pure Chemical Industries LTD, Osaka, Japan) and 0.1% Sodium Dodecyl Sulfate (Sodium Lauryl Sulfate) (SDS) (Wako Pure Chemical Industries LTD, Osaka, Japan) to the culture plate. Fluorescence intensity in this lysate was measured using excitation wavelength of 485 nm and emission wavelength of 583 nm (Fluoroskan, Ascent CE, Labsystems, USA) (Dainippon Pharmaceutical Co., LTD, Tokyo, Japan).
  • the numbers of the BCECF-AM-incorporating cells cultured under the respective conditions was measured, and fluorescence intensity per cell was measured.
  • the actual numbers of the peripheral blood monocytes adhering to the endothelial cells were respectively calculated from the fluorescence intensity obtained in the experiment described above.
  • peripheral blood monocyte adhesion to vascular endothelial cells in the early stage of the onset is important.
  • firm cell-cell adhesion is accomplished by binding of LFA-1 existing in the peripheral blood monocytes to ICAM-1 existing in the vascular endothelial cells, and signals of activation are sent into the cells.
  • the number of the cells adhering to the vascular endothelial cells was low in the peripheral blood monocytes cultured with 100 or 500 ⁇ M spermine for 72 to 80 hours, as compared with that in the peripheral blood monocytes cultured without spermine.
  • the number of the adhering cells was not decreased in the culture with 500 ⁇ M spermine for 20 hours.
  • FIG. 18 a similar result was obtained for the culture with spermidine.
  • the adhesion to the vascular endothelial cells of the peripheral blood monocytes cultured with putrescine was not inhibited. This demonstrated that spermine and spermidine inhibit the peripheral blood monocyte adhesion to the vascular endothelial cells.
  • Peripheral blood monocytes particularly T-cell lymphocyte, monocyte, macrophage and the like have antitumor activity to tumor cells and can kill the tumor cells by this activity. Since T-cell lymphocyte, monocyte, and macrophage are also contained in peripheral blood monocytes, the peripheral blood monocytes have cellular cytotoxicity to tumor cells. It has been known that when lymphokine (interleukin-2), one of proteins called cytokine which is secreted in trace amounts by cells is cultured with the peripheral blood monocytes, the cellular cytotoxicity is increased. Meanwhile, it is known that LFA-1 function is important for the effect of the peripheral blood monocyte.
  • lymphokine interleukin-2
  • peripheral blood monocytes cultured with the polyamines were stimulated with interleukin-2 and examined for their antitumor activity.
  • peripheral blood monocytes For peripheral blood monocytes, RPMI1640 mixed with 10% human serum was used, to which spermine was added in the final concentration of 0 ⁇ M or 100 ⁇ M in the culture medium.
  • the peripheral blood monocytes were cultured in the culture medium containing spermine for 12 to 18 hours and then all recovered from the culture medium.
  • the recovered peripheral blood monocytes were washed three times to remove the culture medium, spermine, and spermidine adhering on the cell surface.
  • the washed cultured peripheral blood monocytes were supplemented with a trace amount (final concentration: 25 U/mL) of a protein which is one kind of cytokine called interleukin-2 (Upstage Biotechnology Inc., Waltham, USA), and cultured in cell culture medium (RPMI1640 containing 10% fetal bovine serum) for 72 hours.
  • Daudi cells Bovine lymphoma cells
  • radioisotope 51 Cr (sodium dichromate); Daiichi Pure Chemicals Co., Ltd) by addition of the radioisotope to the cell and culture at 37° C. for 1 hour.
  • the peripheral blood monocytes cultured with interleukin-2 as well as the Daudi cells were cultured together in the same cell culture plate. Following culture for 3.5 hours, the culture supernatant was recovered, and the amount of 51 Cr in the culture supernatant was measured with a scintillation counter ( ⁇ -counter, LKB).
  • ⁇ -counter ⁇ -counter
  • the cellular cytotoxicity of the peripheral blood monocytes cultured overnight with 100 ⁇ M spermine was reduced. It has been clarified that expression of LFA-1 is most important for exerting cellular cytotoxicity of killer cells in peripheral blood monocytes activated by interleukin-2 (IL-2) (104). It was shown that the LAK activity (cellular cytotoxicity of the cells stimulated with IL-2) of the cells cultured with spermine for 12 to 16 hours is decreased. From this result, the above-described inhibition of LFA-1 function of the peripheral blood monocytes by the polyamines could be recognized with more reliability.
  • IL-2 interleukin-2
  • T-cell lymphocyte contained in peripheral blood monocytes is stimulated on contact with a substance called plant protein lectin (Phytohemagglutinin (hereinafter, PHA) or Concanavalin Agglutinin (Hereinafter, Con-A)), and causes blast transformation.
  • PHA phytohemagglutinin
  • Con-A Concanavalin Agglutinin
  • Peripheral blood monocytes were cultured in cell culture medium (RPMI1640 containing 10% human serum) containing 0 ⁇ M or 100 ⁇ M spermine for 12 to 18 hours. Following the culture, the cells were washed three times with PBS ( ⁇ ) to remove extracellular polyamines. The washed peripheral blood monocytes were cultured for 64 hours in culture medium (RPMI1640 containing 10% fetal bovine serum) mixed with PHA (Difco Laboratories, Detroit, Mich., USA) or Con-A (Sigma chemical co., St. Louis, USA). Then, 3 H-thymidine (Amersham) was added to the cells, followed by additional culture for 8 hours.
  • RPMI1640 containing 10% human serum containing 0 ⁇ M or 100 ⁇ M spermine for 12 to 18 hours. Following the culture, the cells were washed three times with PBS ( ⁇ ) to remove extracellular polyamines. The washed peripheral blood monocytes were cultured for 64 hours in culture medium (RPMI1640
  • peripheral blood monocytes were recovered, and radioactivity of the cells was measured (liquid scintillation counter, LKB-1205, LKB). Since the peripheral blood monocytes activated by mitogen (PHA, Con-A, or the like) cause blast transformation, the cells take up 3 H-thymidine therein. Accordingly, function of cell activation can be measured by measuring the amount of intracellular 3 H-thymidine.
  • This experiment investigated cellular uptake of polyamines in culture medium by cultured peripheral blood monocytes.
  • the peripheral blood monocytes were cultured in culture medium supplemented with 500 ⁇ M putrescine, spermidine, or spermine. Following culture for 16 hours, the cells were recovered from the culture plate and placed into a 50-mL tube. The tube was centrifuged (4° C., 10 min., 100 rpm), and the whole culture medium was aspirated. The cells were washed with 50 mL PBS ( ⁇ ) and centrifuged again, and the supernatant was aspirated. This procedure was repeated three times, and PBS ( ⁇ ) was added to the cells to adjust the cell concentration to approximately 1 ⁇ 10 7 cells/mL, followed by freezing to ⁇ 20° C.
  • FIG. 20 shows polyamine concentration per 1 ⁇ 10 7 cells/mL of the cell suspension obtained by suspending the peripheral blood monocytes cultured with in the culture medium supplemented with 500 ⁇ M each of the polyamines for 16 hours. Only the concentration of the cultured polyamine was increased in the peripheral blood monocytes cultured with each polyamine. As described above, the unit of the extracellular polyamines was supposed to be pM, whereas the unit of the measured polyamine concentration of the cell suspension was ⁇ M. Accordingly, it is considered that the polyamines contained in the culture medium exert little influence on concentration measurement. Since the measured cell suspension was the one in which the cells were disrupted by freezing to leak the intracellular polyamines into the suspension, it is considered that the measurement value reflects polyamine concentration in the cells. Thus, it is obvious that the polyamines in the culture medium are taken up into the peripheral blood monocytes.
  • spermine and spermidine contained in cells inhibit the expression of CD11a and CD18 on the surface of peripheral blood monocytes (lymphocyte, monocyte, and macrophage) which are immunocytes in human blood and inhibit the function of the cell surface molecule LFA-1 composed of these two molecules. This inhibition is selective and is not exerted on other cell membrane antigen molecules, most of which are rather promoted. The general index of cell function is also enhanced.
  • the present invention can be practiced easily and provides quite useful selective inhibitors of LFA-1.
  • the inhibitors of the present invention can be expected to have sufficient effects as agents for the treatment or inhibition of these diseases.
  • LFA-1 plays an important role in the pathology of each disease of autoimmune disease (type I diabetes (insulin-dependent diabetes mellitus), Graves' disease (Basedow disease), Hashimoto disease, autoimmune arthritis (Lyme arthritis, chronic rheumatoid arthritis), autoimmune cerebrospinal peripheral neuritis or degeneration, Sjogren's syndrome, uveitis, retinitis or degeneration, autoimmune renal disease (glomerulonephritis and the like), inflammatory bowel disease (Crohn disease, ulcerative colitis and the like), and primary cholangitis), allergic disease, ischemic reperfusion injury, and diabetic retinopathy.
  • autoimmune disease type I diabetes (insulin-dependent diabetes mellitus), Graves' disease (Basedow disease), Hashimoto disease, autoimmune arthritis (Lyme arthritis, chronic rheumatoid arthritis), autoimmune cerebrospinal peripheral neuritis or degeneration, Sjogren's syndrome, uveitis, retinitis
  • an amount of the polyamines ingested in a day in adult is about 350 to 550 ⁇ mol from an amount of the polyamines contained in foods (105) and average meal in a day (106).
  • spermine has toxicity when contained at 0.2% or more in meal but produces good effect when contained in concentration that is an order of magnitude smaller, and orally administered spermidine produces good effect at 0.05% (108).
  • Til et al. have reported that in the acute toxicity test using rats, 50% lethal dose (LD50) of spermidine and spermine is 600 mg/kg of body weight. They have also reported that in the subacute toxicity test (administration for 6 weeks), no side effect is observed in spermidine administered in an amount up to 83 mg/kg of body weight/day and spermine administered in an amount up to 19 mg/kg of body weight/day. Furthermore, the scope of the study has clarified not only the dose expressing acute toxicity but also the concentration of the substances taken internally as described above. Moreover, both spermine and spermidine have water absorbency and are stable in the form of aqueous solution. As described above, it has been clarified not only that these substances are absorbed in their original forms from intestine and carried to each tissue in the body but also that polyamine concentration in peripheral blood monocyte is also increased.
  • spermine and spermidine are 202.34 and 145.24, respectively.
  • 5 mmol spermine and spermidine correspond to approximately 1,012 mg and 726 mg, respectively. This corresponds to 20.23 mg of spermine/kg of body weight and 14.5 mg of spermidine/kg of body weight, and both spermidine and spermine provided in these amounts are said to be safe.
  • These figures are those for theoretically exerting sufficient inhibition effect on LFA-1 in single administration. However, spermine and spermidine gradually exert inhibition effect on LFA-1 after taken up into cells. Therefore, consecutive administration of them in small amounts is more realistic.
  • This amount corresponds to approximately 988 ⁇ mol spermine and 1,377 ⁇ mol spermidine.
  • the amounts are sufficiently reasonable from a safety standpoint.
  • the amounts are considered to be amounts capable of exerting sufficient treatment effects.
  • smaller amounts of the polyamines are preferably administered.
  • the upper limit per kg of body weight at one dose or daily dose is given at up to 200 ⁇ mol in an aqueous or alcohol solution having any one or both of spermine and spermidine concentrations ranging from 0.1% to 0.001%.
  • spermine and spermidine among polyamines were mainly used, because they are typical biogenic polyamines. It will be evident that other polyamines have similar effects.

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US20080064105A1 (en) * 2004-10-29 2008-03-13 Baxter Healthcare Corporation Animal protein-free media for cultivation of cells
WO2010081862A3 (fr) * 2009-01-14 2010-10-07 Katholieke Universiteit Leuven, K.U. Leuven R&D Méthodes et préparations pour la protection de patients en état critique
EP2209371A4 (fr) * 2007-10-19 2011-11-02 Compositions et procédés pour le traitement de la rétinopathie diabétique
WO2012151554A1 (fr) * 2011-05-04 2012-11-08 President And Fellows Of Harvard College Polyamines utilisables en vue du traitement de biofilms
US9216174B2 (en) 2003-11-05 2015-12-22 Sarcode Bioscience Inc. Modulators of cellular adhesion
EP3052189A4 (fr) * 2013-10-03 2017-11-01 Leuvas Therapeutics Modulation de l'activité leucocytaire dans le traitement d'une maladie dégénérative neuro-inflammatoire

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WO2009093454A1 (fr) * 2008-01-23 2009-07-30 Jichi Medical University Composition alimentaire, composition d'aliments comprenant la composition alimentaire, et procédé pour alimenter des animaux à l'aide de la composition d'aliments
US20180283347A1 (en) * 2015-10-08 2018-10-04 Menash ELI-NOVAK System for converting kinetic energy of ocean waves into electrical energy
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US9216174B2 (en) 2003-11-05 2015-12-22 Sarcode Bioscience Inc. Modulators of cellular adhesion
US9248126B2 (en) 2003-11-05 2016-02-02 Sarcode Bioscience Inc. Modulators of cellular adhesion
US9809796B2 (en) 2004-10-29 2017-11-07 Baxalta GmbH Animal protein-free media for cultivation of cells
US10655099B2 (en) 2004-10-29 2020-05-19 Baxalta Incorporated Animal protein-free media for cultivation of cells
US20110081680A1 (en) * 2004-10-29 2011-04-07 Baxter International Inc. Animal Protein-Free Media For Cultivation of Cells
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US20080064105A1 (en) * 2004-10-29 2008-03-13 Baxter Healthcare Corporation Animal protein-free media for cultivation of cells
US10138461B2 (en) 2004-10-29 2018-11-27 Baxalta GmbH Animal protein-free media for cultivation of cells
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US8748156B2 (en) 2004-10-29 2014-06-10 Baxter International Inc. Animal protein-free media for cultivation of cells
US9222075B2 (en) 2004-10-29 2015-12-29 Baxalta Incorporated Animal protein-free media for cultivation of cells
US9714411B2 (en) 2004-10-29 2017-07-25 Baxalta GmbH Animal protein-free media for cultivation of cells
EP2209371A4 (fr) * 2007-10-19 2011-11-02 Compositions et procédés pour le traitement de la rétinopathie diabétique
US10960087B2 (en) 2007-10-19 2021-03-30 Novartis Ag Compositions and methods for treatment of diabetic retinopathy
WO2010081862A3 (fr) * 2009-01-14 2010-10-07 Katholieke Universiteit Leuven, K.U. Leuven R&D Méthodes et préparations pour la protection de patients en état critique
WO2012151554A1 (fr) * 2011-05-04 2012-11-08 President And Fellows Of Harvard College Polyamines utilisables en vue du traitement de biofilms
EP3052189A4 (fr) * 2013-10-03 2017-11-01 Leuvas Therapeutics Modulation de l'activité leucocytaire dans le traitement d'une maladie dégénérative neuro-inflammatoire

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